Methods and compositions for improving growth of meat-type poultry

ABSTRACT

The present invention provides methods of improving growth performance, improving the efficiency of feed utilization, increasing feed digestibility, and decreasing mortality of immature and developing animals receiving animal feed. Methods of producing a crude keratinise enzyme extract and animal feed supplements for achieving the same are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/402,228 filed Aug. 9, 2002, the disclosure of which isincorporated herein by reference in its entirety.

STATEMENT OF FEDERAL SUPPORT

Research directed to this invention is supported in part by USDepartment of Agriculture Small Business Innovation Research Grant No.2002-33610-11850. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods of improving growth performanceof immature and developing animals receiving animal feed and animal feedsupplements for achieving the same.

BACKGROUND OF THE INVENTION

Broiler chick starter diets contain a considerable amount of crudeprotein. Most of the crude protein is obtained from traditional feedingredients such as soybean meal. Approximately 90% of the crude proteinpresent in soybean meal (48% crude protein content) is highly digestiblefor poultry (National Research Council (1994). Nutrient requirements ofpoultry. 9^(th) revised Ed. National Academy Press, Washington, D.C.).Although the traditional corn-soybean meal broiler starter diets areconsidered highly digestible, they often contain a variety of complexproteins that are not easily digested by a young chick due to the lackof necessary innate enzymes at early stages of life (Uni, et al. (1999)Poultry Sci. 78: 215-222). Inclusion of proteases in broiler diets hasbeen suggested, but much of the early work with protease addition tocereal grain-based diets did not result in any improvements in birdperformance (Jensen, et al. (1957) Poultry Sci. 36: 919-921).

More recently, enzyme supplementation of poultry diets with enzymemixtures, including proteases and amylases, has produced someimprovements in growth performance (Greenwood, et al. (2002) PoultrySci. 81(Suppl. 1): 25; Burrows, et al. (2002) Poultry Sci. 81(Suppl. 1):29; Short, et al. (2002) Poultry Sci. 81(Suppl. 1): 136). Supplementinga corn-soybean broiler starter diet with an enzyme preparationcontaining a mixture of xylanase, protease, and amylase resulted inimprovements in body weight at 14 and 42 days of age with no significanteffects on feed conversion ratio (Greenwood, et al. (2002) supra). Uponsupplementing corn-soy based duck diets with the same enzyme mixture,the enzyme supplementation resulted in improvements in body weight gainand feed conversion ratio (Burrows, et al. (2002) supra).

Poultry feed further contains some complex antinutritional and/orindigestible compounds. Some of these compounds, such as the nonstarchpolysaccharides, absorb water into a viscous mass within the chyme fromwhich nutrients are not readily absorbed (Odetallah, 2000; Odetallah, etal. (2002) supra). As chyme viscosity increases, the rate of diffusionof digestive enzymes and nutrients decreases, thus impeding nutrientabsorption by the enterocyte. Fat micelle formation and absorption alsodecrease as chyme viscosity increases, thus impairing absorption of manyof the fat-soluble compounds, including fat-soluble vitamins, pigments,and lipids (Ferket and Veldkamp (1999) In; Proceedings of the 1998 WorldPoultry Science Association, pgs 43-52). Therefore, viscosity reductionachieved by endolytic enzyme activity may play a role in the improvementseen in young chicks fed high-viscosity cereals, and the relativeeffectiveness of various enzymes appears to be related to theirviscosity-reducing capability (Rotter, et al. (1990) J. Sci. Food Agric.50:19-27).

PWD-1 keratinase is an enzyme that was originally purified from thegrowth medium of Bacillus licheniformis PWD-1 (Williams, et al. (1990)Appl. Environ. Microbiol. 56:1509-1515; Lin, et al. (1992) Appl.Environ. Microbiol. 58:3271-3275). PWD-1 keratinase hydrolyzes a broadrange of protein substrates including casein, collagen, elastin andkeratin (Shih (2001) In: Proceedings International Conference ofAgricultural Science and Technology, Beijing, China, pgs 244-247). PWD-1keratinase has been used to produce hydrolyzed feather meal byincubating commercial feather meal with cell-free keratinase overnight(Carter (1998) Bacterial Keratinase: Assay development and nutritionalapplication. Ph.D. Thesis, North Carolina State University, Raleigh,N.C.). See also U.S. Pat. Nos. 4,908,220; 5,186,961; and 5,063,161 toShih et al.

In spite of the foregoing, there remains a need for additional methodsof enhancing growth performance of broiler chicks and animal feedsupplements that accomplish the same.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions that enhancegrowth performance of immature and developing animals receiving animalfeed.

An aspect of the invention relates to a method of growing meat-typepoultry comprising feeding meat-type poultry a corn-soybean meal feed asa poultry diet wherein the feed further comprises keratinase in anamount effective to enhance the weight gain of meat-type poultry.

Another aspect of the invention relates to a method of growing meat-typepoultry comprising feeding meat-type poultry a corn-soybean meal feed asa starter diet wherein the feed further comprises keratinase in anamount effective to enhance the weight gain of the meat-type poultry.

A further aspect of the invention relates to a method of improving theefficiency of feed utilization of an animal feed in meat-type poultrycomprising feeding meat-type poultry a corn-soybean meal feed as apoultry diet wherein the feed further comprises keratinase in an amounteffective to improve the efficiency of feed utilization of an animalfeed in meat-type poultry.

An additional aspect of the present invention relates to a method ofincreasing the digestibility of an animal feed in meat-type poultrycomprising feeding meat-type poultry a corn-soybean meal feed as apoultry diet wherein the feed further comprises keratinase an amounteffective to increase the digestibility of an animal feed in meat-typepoultry.

Another aspect of the invention relates to a method of reducingmortality in meat-type poultry comprising feeding meat-type poultry acorn-soybean meal feed as a starter diet wherein the feed furthercomprises keratinase in an amount effective to reduce mortality ofmeat-type poultry.

A further aspect of the invention relates to an animal feed consistingessentially of keratinase, protein, and carbohydrate.

An additional aspect of the invention relates to a method of producingcrude keratinase enzyme extract.

The present invention further relates to improving the nutritionalstatus of a hatchling, and thereby increasing disease resistance andsurvivability of the immature bird to achieve a greater level of growthperformance meat-type poultry.

DETAILED DESCRIPTION OF THE INVENTION

The foregoing and other aspects of the present invention will now bedescribed in more detail with respect to other embodiments describedherein. It should be appreciated that the invention can be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

All publications, U.S. patent applications, U.S. patents and otherreferences cited herein are incorporated by reference in theirentireties.

As used herein, the term “meat-type poultry” refers to any avian speciesthat is produced or used for meat consumption as understood by oneskilled in the art. Examples of such avian species include, but are notlimited to, chickens, turkeys, ducks, geese, quail, pheasant, ratites,and the like.

As used herein, the term “immature bird” refers to a member of the avianspecies that lacks complete growth, differentiation, or development.Such members can have the potential capacity to attain a definite matureform or state. An immature bird can be from about 1 to about 50 daysold, preferably about 1 to about 21 days old, and more preferably about1 to about 5 days old, or can have a body weight comparable to birdswithin these ranges.

As used herein, the term “developing bird” refers to a member of theavian species that is older or weighs more than an immature bird.

As used herein, the term “mature bird” refers to a member of the avianspecies that is older or weighs more than a developing bird.

As used herein, the term “broiler chick” refers to any immature chickenproduced or eventually used for meat consumption.

As used herein, the term “poultry diet” refers to a diet that can beadministered to a member of the avian species to promote and maintaingrowth of the bird. A poultry diet can contain sources of protein,vitamins, minerals, energy such as fat, carbohydrates, and additionalprotein, antibiotics, and other substances or compounds known to beincluded in animal feeds, in particular, poultry feeds. Poultry diet isinclusive of, but not limited to, a starter diet, a grower-type diet,and a finisher-type diet. A “starter diet” refers to a diet that can beadministered to an animal starting from birth or hatch until a desiredage and/or weight is obtained. A “grower-type diet” refers to a dietthat can be administered to an animal upon completion of the startergrowth phase. A “finisher-type diet” refers to a diet that can beadministered to an animal during the period of development through thetime of slaughter.

As used herein, the terms “growth” or “growth performance” refer toincreases in either, or both, weight and size (e.g., height, width,diameter, circumference, etc.) over that which would otherwise occurwithout implementation of the methods and/or administration of thecompositions of the present invention. Growth can refer to an increasein the mass (e.g., weight or size) of the entire animal or of aparticular tissue (e.g., muscle tissue in general or a specific muscle).Alternatively, growth can indicate a relative increase in the mass ofone tissue in relation to another, in particular, an increase in muscletissue relative to other tissues (e.g., adipose tissue). Growth furtherrelates to nutritional status and disease resistance wherein improvementof nutritional status and/or increase in disease resistance is alsoindicative of improved growth performance.

In view of the foregoing, embodiments according to the present inventionrelate to methods of growing meat-type poultry, comprising feedingmeat-type poultry an animal feed poultry diet wherein the feed furthercomprises keratinase and is added to the poultry diet in an amounteffective to enhance the weight gain of the meat-type poultry. Thepoultry diet can be an animal feed which includes sources of protein,for example, soybean meal, fish meal, blood meal, poultry by-product(ground poultry offal), meat meal, wheat-meal, rapeseed, canola andcombinations of the same. The animal feed further includescarbohydrates, for example, corn, oats, barley, sorghum, or combinationsof the same that can be ground into a meal for use in the animal feed.Additionally, the animal feed can include vitamins, minerals, fat,antibiotics, and other substances or compounds as necessary or desired.Non-limiting examples of animal feed poultry diets include cereal-basedfeeds including cereals such as barley, corn, soya, wheat, triticale,and rye. Corn-soybean, wheat-soybean, and wheat-corn-soybean,sorghum-soybean, and corn-sorghum-soybean represent other non-limitingexamples of suitable animal feeds according to the present invention.When the poultry diet is a corn-soybean meal feed, the corn-soybean mealfeed comprises from about 60 to about 70% corn by weight and from about20 to about 30% soybean by weight.

The poultry diet can further be categorized as a starter diet, agrower-type diet, or a finisher-type diet. The precise composition andphysical characteristics of the animal feed, and thus the poultry diet,will depend upon the species for which the feed is intended, the ageand/or weight of the animal, and the duration of feeding, and can bereadily determined by those skilled in the art.

According to embodiments of the present invention, the methods ofgrowing meat-type poultry do not require concurrently providing aspecific keratin-containing substrate along with the keratinase; Forexample, in embodiments of the present invention, the keratinase candirectly supplement a poultry diet as a feed additive in contrast toproducing a hydrolyzed feather meal as described in Carter, 1998, Thus,the animal feed can be essentially free of keratin (e.g., not more than1 or 2% by weight keratin.)

A keratinase suitable for practicing the present invention is obtainedfrom Bacillus licheniformis strain PWD-1, which is described in U.S.Pat. Nos. 4,908,220 and 4,959,311 (the disclosures of all patentreferences cited herein are to be incorporated herein by reference).This bacterium was deposited with the American Type Culture Collection(ATCC) in Rockville, Md., USA in accordance with the Budapest Treaty onMar. 23, 1988, and assigned ATCC Accession No. 53757. Other keratinasesthat can be used to practice the present invention are available from avariety of bacterial sources, such as Streptomyces fradiae. Seegenerally U.S. Pat. No. 2,988,487 to Nickerson; See also Goktan, D.,“Decomposition Rates of Keratinous Material Used by CertainMicroorganisms,” (Abstract No. 207369b), Microbial Biochem. 101, 333(1984); Daniels, G., “The Digestion of Human Hair Keratin by MicrosporumCanis,” J. Gen. Microbiol. 8, 289 (1953); Koh, W. et al., “KeratinolyticEnzymes from Aspergillus flavus and Aspergillus niger,” Bacillus. Aust.J. Biol. Sci. 274 (1959); Molyneaux, G. S., “The Digestion of Wool by aKeratinolytic Bacillus,” Aust. J. Biol. Sci. 274 (1959); Noval, J. andNickerson, W., “Decomposition of Native Keratin by StreptomycesFradiae,” J. Bacteriol. 77, 251 (1959); Kapica, L. and Blank, F.,“Growth of Candida Parapsilosis with Keratin as Sole Source ofNitrogen,” Dermatologica 117, 433 (1958); Kapica, L. and Blank, F.,“Growth of Albicans on Keratin as Sole Source of Nitrogen,”Dermatologica 115, 81 (1957).

Keratinase for practicing the present invention can be obtained bygrowing a host cell which contains nucleic acid sequences encoding akeratinase, under conditions which permit expression of the encodedkeratinase, filtering the medium to remove the cells and collecting andconcentrating the remaining supernatant by ultrafiltration to obtain thekeratinase. Beneficiary co-factor(s) can also be obtained.

While strains of B. licheniformis are exemplified herein, it iscontemplated that other eukaryotic and prokaryotic microbes containingnucleic acid sequences encoding a keratinase may also be useful inproducing an animal feed supplement of the present invention. Eukaryoticand prokaryotic microbes containing nucleic acid sequences encoding akeratinase may include those which naturally produce the enzyme as wellas strains genetically modified to express keratinase. In general,recombinant production of a protein may require incorporation of nucleicacid sequences encoding said protein into a recombinant expressionvector in a form suitable for expression of the protein in a host cell.A suitable form for expression provides that the recombinant expressionvector includes one or more regulatory sequences operatively-linked tothe nucleic acids encoding the a keratinase protein in a manner whichallows for transcription of the nucleic acids into mRNA and translationof the mRNA into the protein. Regulatory sequences may includepromoters, enhancers and other expression control elements (e.g.,polyadenylation signals). Such regulatory sequences are known to thoseskilled in the art and are described in Goeddel D. D., ed., GeneExpression Technology, Academic Press, San Diego, Calif. (1991). Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transfected and/orthe level of expression required. Nucleic acid sequences or expressionvectors harboring nucleic acid sequences encoding a keratinase proteinmay be introduced into a host cell, which may be of eukaryotic orprokaryotic origin, by standard techniques for transforming cells.Suitable methods for transforming host cells may be found in Sambrook,et al. (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold SpringHarbor Laboratory Press (2000)) and other laboratory manuals. The numberof host cells transformed with a nucleic acid sequence encoding akeratinase protein will depend, at least in part, upon the type ofrecombinant expression vector used and the type of transformationtechnique used. Nucleic acids may be introduced into a host celltransiently, or more typically, for long-term expression of a keratinaseprotein the nucleic acid sequence is stably integrated into the genomeof the host cell or remains as a stable episome in the host cell. Onceproduced, a keratinase protein may be recovered from culture medium as asecreted polypeptide, although it also may be recovered from host celllysates when directly expressed without a secretory signal.

Eukaryotic microbes such as yeast cultures may be transformed withvectors carrying nucleic acid sequences encoding a keratinase. See,e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the mostcommonly used among lower eukaryotic host microorganisms, although anumber of other strains are commonly available. Yeast vectors maycontain an origin of replication from the 2 micron yeast plasmid or anautonomously replicating sequence (ARS), a promoter, DNA encoding akeratinase such as that provided in U.S. Pat. No. 5,712,147, sequencesfor polyadenylation, and transcription termination, and a selectiongene. An exemplary plasmid is YRp7, (Stinchcomb, et al. (1979) Nature282:39; Kingsman, et al. (1979) Gene 7:141; Tschemper, et al. (1980)Gene 10:157). Suitable promoting sequences in yeast vectors include thepromoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman, etal. (1980) J. Biol. Chem. 255:2073) or other glycolytic enzymes (Hess,et al. (1968) J. Adv. Enzyme Reg. 7:149; Holland, et al. (1978)Biochemistry 17:4900). Suitable vectors and promoters for use in yeastexpression are further described in EPO Publication. No. 73,657.Further, fungal strains such as of Trichoderma (e.g., T.longibrachiatum, T. reesei or T. viride) are particularly useful inexpressing secreted enzymes.

Prokaryote host cells which may be used to produce a keratinase includegram negative or gram-positive organisms, for example Escherichia coli(E. coli) or Bacilli. Exemplary host cells are E. coli W3110 (ATCC27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC31,446). A broad variety of suitable prokaryotic and microbial vectorsare available. E. coli is typically transformed using pBR322. Promotersmost commonly used in recombinant microbial expression vectors includethe beta-lactamase (penicillinase) and lactose promoter systems (Chang,et al. (1978) Nature 275:615; Goeddel, et al. (1979) Nature 281:544), atryptophan (trp) promoter system (Goeddel, et al. (1980) Nucleic AcidsRes. 8:4057; EPO Publication No. 36,776) and the tac promoter (De Boer,et al. (1983) Proc. Natl. Acad. Sci. USA 80:21). The promoter andShine-Delgarno sequence (for prokaryotic host expression) are operablelinked to the DNA encoding the keratinase, i.e., they are positioned soas to promote transcription of keratinase messenger RNA from the DNA. Aspecies of Bacillus is preferably used in the production of akeratinase. Recombinant expression vectors for Bacillus are well knownto those of skill in the art. Bacillus strains can be B. alkalophilus,B. amyloliquefaciens, B. brevis, B. circulans, B. coagulans, B. firmus,B. lautus, B. lentus, B. licheniformis, B. megaterium, B. pumilus, B.stearothermophilus, B. subtilis, and B. thuringiensis. In a preferredembodiment, strains of B. licheniformis are utilized. In someembodiments, B. licheniformis strains T399D or PWD-1 are utilized.

As provided herein, a keratinase enzyme may be produced by culturing ahost cell as described above under conditions that permit expression ofthe encoded keratinase, and collecting the expressed keratinase. Thehost cell may be cultured under conditions in which the cell grows, andthen cultured under conditions which cause the expression of the encodedkeratinase, or the cells may be caused to grow and express the encodedkeratinase at the same time. Such conditions are well known to one ofskill in the art and may vary with the host cell and the amount ofenzyme expression level desired.

In some embodiments, the medium used to cultivate the transformed hostcells may be any medium suitable for keratinase production. Thekeratinase is recovered from the medium by conventional techniquesincluding separation of the cells from the medium by centrifugation, orfiltration, and concentration of the proteins in the supernatant orfiltrate by ultrafiltration or evaporation followed by drying vialyophilization or spray-drying.

Alternatively, the culture supernatant may be spray-dried or lyophilizedafter separation without being concentrated.

The keratinase should be present in an amount at least sufficient toachieve the intended effect, but the upper limit to the amount ofkeratinase can be determined based upon achieving the intended effect.In some embodiments, the animal feed comprises from about 0.01% to about20% Bacillus licheniformis PWD-1 keratinase by weight. Additionally,keratinases used in practicing the present invention can be in crudeform or in pure form. Keratinases in crude form can be prepared, forexample, by separating bacterial cells which produce the keratinase fromtheir liquid growth media, the liquid growth media comprising crudekeratinase. Alternatively, the cells can be lysed (chemically orphysically) in a liquid growth media to produce a crude, cell freeextract. Other means of preparing such an extract will be apparent topersons skilled in the art. The crude keratinase can be included in thefeed in any form compatible therewith, such as in an aqueous form or inlyophilized form. In some embodiments, the crude keratinase is in thelyophilized form.

Pure (or substantially pure) keratinases can be obtained by separatingthe crude keratinase described above into its individual constituents,in accordance with known techniques. See generally W. Jakoby, Ed.,Enzyme purification and Related Techniques, Methods in Enzymology, vol.22 (1971) and vol. 104, pt. C (1984), Academic Press, NY. Numeroussuitable separation procedures, such as column chromatography, are knownto persons skilled in the art. The individual constituent proteins canbe screened for their ability to degrade keratinaceous material, andthat constituent which best degrades keratinaceous material comprisesthe keratinase. Like the crude keratinase, the pure keratinase can beemployed in any suitable form, including aqueous form and lyophilizedform.

Embodiments of the present invention further relate to methods ofimproving the efficiency of feed utilization of an animal feed inmeat-type poultry comprising feeding meat-type poultry an animal feedpoultry diet wherein the feed further comprises keratinase in an amounteffective to improve the efficiency of feed utilization of an animalfeed provided to meat-type poultry. The animal feed can include theanimal feeds as described above and, in particular embodiments can becorn-soybean meal. The keratinase can include keratinases as describedabove including, but not limited to, Bacillus licheniformis PWD-1keratinase. As described above, the keratinase can be a crude extract orpure form enzyme.

Improving the efficiency of feed utilization refers to a reduction inthe Feed Conversion Ratio (FCR) as compared with that which wouldotherwise occur without implementation of the methods and/oradministration of the compositions of the present invention. The FCR isthe ratio of the amount of feed consumed relative to the weight gain ofan animal. In one embodiment of the present invention, the improvedefficiency of feed utilization can occur by increasing gastrointestinalnutrient absorption without a concomitant increase in intestinal energyexpenditure. In another embodiment of the present invention, theimproved efficiency of feed utilization can occur by increasing thedigestibility of the animal feed. In another embodiment of the presentinvention, the improved efficiency of feed utilization can occur bydecreasing the viscosity of the animal feed. In particular embodiments,the present invention relates to methods of increasing the digestibilityof an animal feed in a meat-type poultry comprising feeding meat-typepoultry an animal feed poultry diet wherein the feed further comprisesBacillus licheniformis PWD-1 keratinase in an amount effective toincrease the digestibility of an animal feed in meat-type poultry. Theanimal feed can include the animal feeds as described above and, inparticular embodiments, can be corn-soybean meal. The keratinase caninclude keratinases as described above including, but not limited to,Bacillus licheniformis PWD-1 keratinase. As described above, thekeratinase can be a crude extract or pure form enzyme. Increasing thedigestibility of an animal feed refers to increasing the availability ofnutrients absorbed from the animal's gut without a concurrent increasein feed intake or nutrient ingestion. In some embodiments of the presentinvention, the viscosity of materials present in the animal's gut ordigesta viscosity is reduced. In other embodiments, the entrapment ofnutrients rendering them nutritionally unavailable to the animal isreduced.

In other embodiments, the present invention relates to methods ofreducing mortality in meat-type poultry comprising feeding meat-typepoultry an animal feed poultry diet wherein the feed further comprises akeratinase in an amount effective to reduce the mortality of meat-typepoultry, for example immature birds, and more specifically, broilerchicks. The animal feed can include the animal feeds as described aboveand, in particular embodiments, can be corn-soybean meal. The keratinasecan include keratinases as described above including, but not limitedto, Bacillus licheniformis PWD-1 keratinase. As described above, thekeratinase can be a crude extract or pure form enzyme. Reducingmortality refers to increasing the survivability or decreasing the deathrate in animals after birth or hatch as compared with that which wouldotherwise occur in the absence of implementation of the methods and/oradministration of the compositions of the present invention. Mortalitycan be from any cause, in particular, stress, stunting, “starveouts”,and disease. In some embodiments, the present invention reducesmortality in immature birds. In other embodiments, the birds are about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, or 35 days old,preferably about 1 to about 21 days old, and more preferably about 1 toabout 5 days old.

In some embodiments, the present invention relates to an animal feedcomprising protein, carbohydrate, and keratinase as the majorcomponents. The keratinase is a major component supplementing the animalfeed. The animal feed can include the animal feeds as described aboveand, in particular embodiments, can be corn-soybean meal. The keratinasecan include keratinases as described above including, but not limitedto, Bacillus licheniformis PWD-1 keratinase. As described above, thekeratinase can be a crude extract or pure form enzyme.

The animal feed supplement provided by the present invention can bemixed directly with the animal feed, such as one comprising barley, toprepare the final feed. Alternatively, the animal feed supplement can bemixed with one or more other animal feed supplements such as a vitaminanimal feed supplement, a mineral animal feed supplement and an aminoacid animal feed supplement. The resulting animal feed supplementincluding several different types of components may then be mixed in anappropriate amount with the animal feed.

The animal feed of the present invention comprises keratinase in anamount at least sufficient to achieve the intended effect, wherein theupper limit to the amount of keratinase can be determined based uponachieving the intended effect. Intended effects include, but are notlimited to, enhancing animal growth performance, such as weight gain,improving the efficiency of feed utilization, increasing feeddigestibility, and decreasing mortality. The animal feed supplementadded to the animal feed can comprise up to 100% keratinase by weight.The animal feed comprising the supplement comprises from about 5% toabout 25% keratinase by weight. In some embodiments, the keratinase isBacillus licheniformis PWD-1 keratinase.

Any animal is a suitable subject for the present invention, includingcows, sheep, pigs, cats, dogs, ferrets, and avians, however, the presentinvention is preferably employed with monogastric animals. Suitablesubjects can be of any age range including neonatal animals, developinganimals, and mature animals. In some embodiments, the suitable subjectcan be an avian, preferably a chicken, and more preferably a broilerchick. In other embodiments the suitable subject can be a chicken. Instill other embodiments, the suitable subject can be an immature,developing, or mature bird. In other embodiments, the suitable subjectcan be a chicken that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 days old,or within any range of these numbers. Thus, the present inventionprovides a variety of different feeds, including pet feed, poultry feed,and pig feed.

The animal feed supplement of the present invention can also enable aconventional animal feed to be modified by reducing its energy, and/orprotein, and/or amino acid content while simultaneously maintaining thesame nutritional levels of energy, protein, and amino acids available tothe animal. Consequently, the amounts of costly energy and proteinsupplements typically included in an animal feed can be reduced ascompared to conventional feeds.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLE 1 Production of Keratinase from Recombinant B. licheniformisPWD-1 Strain

A fermentation scale-up strategy was designed for the production ofkeratinase, using the wild-type B. licheniformis strain PWD-1.

Flask Culture in LB Medium. Flask culture was carried out inLuria-Bertani (LB) medium that was prepared according to themanufacturer's specification, containing: 1.0 L of distilled water, 15 gBACTO® agar, 10 g NaCl, 10 g BACTO® tryptone, and 5.0 g yeast extract.B. licheniformis strain PWD-1 was streaked from a glycerol stock onto anLB plate and grown at 50° C. for 8-12 hours. A single colony of B.licheniformis strain PWD-1 was then transferred from the LB plate into aflask that contained 500 ml LB medium, and grown at 50° C. for 6 hours.

Seed Cultures. Seed cultures for B. licheniformis strain PWD-1 wereconducted in a medium containing: 0.7 g/L KH₂PO₄, 1.4 g/L K₂HPO₄, 0.1g/L MgSO₄.7H₂O, 10 g/L defatted soy flour, and 0.1 g/L antifoamingagent. The initial pH of the seed culture was adjusted to 7.0 by adding1M HCl or NaOH.

The 500 ml flask culture was transferred into a first stage seedfermentor of about 10 L to 20 L that contained the seed culture medium,and was grown therein at 50° C. for 8-12 hours to reach 2.5% to 5%inoculum size. The first stage seed culture was then transferred to asecond stage seed fermentor of 100 L, 250 L or 800 L, and was growntherein at 50° C. for 8 hours and then shifted to 37° C.

For the seed culture, the cell density reached at least 3×10⁸ CFU/mL atabout 8 or 10 hours of the culture process.

Production Media. The production culture medium used for B.licheniformis strain PWD-1 contained 0.7 g/L KH₂PO₄, 1.4 g/L K₂HPO₄, 0.1g/L MgSO₄.7H₂O, 10 g/L defatted soy flour and 0.1 g/L antifoaming agent.The initial pH of the production culture was adjusted to 7.0 by adding1M HCl or NaOH.

The second stage seed culture was transferred to a production fermentorthat contained the production culture medium for final stage culturing.The final stage culture was carried out at 50° C. for 8 hours, reachinga total culturing time of about 24 to 30 hours before harvesting.

During the above culturing steps, the initial pH of the culture mediumwas adjusted to 7.0, but no pH control during the culture process wasprovided. The optimal level of dissolved oxygen was about 20% for B.licheniformis strain PWD-1. The inoculum size was about 2.5 to 5%, andthe inoculum age was about 8-12 hours.

For the production culture, the peak cell density reached 1.2×10⁹ CFU/mLat about 20 or 24 hours of the culture process. The peak enzymeactivity, as measured by azocasein assay, reached 35-40 A₄₅₀ per mL atabout 24 to 30 hours of the culture process. The pH value of theproduction culture medium changed from 7.0 to 8.3, but the enzymeactivity and productivity stayed at high levels, which indicated that nopH control was necessary.

Recovery and Downstream Processing. The enzyme activity in theproduction culture was checked before harvesting. The culturesupernatant was separated from the cell mass via centrifuge, and thenconcentrated via ultrafiltration or evaporation. The concentrated liquidenzyme was then spray-dried.

Alternatively, the culture supernatant was directly spray-dried afterseparation from the cell mass, without being concentrated.

Enzyme Yield and Enzyme Activity. For 100 L production culture, theenzyme activity measured by azocasein assay before harvesting was 3,000to 3,500 U/mL, and the cell number was 1.3×10⁹ CFU/mL. The total dryweight of the 100 L production culture was 9.12 g/L, including 2.15 g/Linsoluble dry weight and 6.88 g/L soluble dry weight.

The crude enzyme yield was about 1.75-2.0 g/L. The crude enzyme wasprepared by concentration of the fermentation supernatant via Pelliconfiltration with a 5 kDa molecular weight cut-off, and then freeze dried.The enzyme activity of the crude dry enzyme was about 1,000,000 to about1,400,000 U/g, as measured by the azocasein assay. The total proteincontent of the crude dry enzyme was about 30-36%, of which approximately14-20% consisted of pure keratinase.

EXAMPLE 2 Production of Keratinase from Recombinant B. licheniformisT399D Strain

A fermentation scale-up strategy was designed for the production ofkeratinase, using a recombinant Bacillus licheniformis T399D strain(hereinafter the “Bacillus licheniformis strain T1”).

Flask Culture in LB Medium. Flask culture was carried out in LB mediumthat was prepared according to the manufacturer's specification,containing: 1.0 L of distilled water, 15 g BACTO® agar, 10 g NaCl, 10 gBACTO® tryptone, and 5.0 g yeast extract. B. licheniformis strain T1 wasstreaked from a glycerol stock onto LB plates and grown at 37° C. for 18hours. A single colony of B. licheniformis strain T1 was thentransferred from the LB plate into a flask that contained 500 mL LBmedium, and grown at 37° C. for 6 hours. Cell growth was monitored bymeasuring the optical density at 660 nm (Beckman DU Series 660Spectrophotometer, Fullerton, Calif.). After 6 hours of growth, theOD₆₆₀ measured above 1.0.

Seed Cultures. Seed cultures for B. licheniformis strain T1 were grownin a medium containing: 0.7 g/L KH₂PO₄, 1.4 g/L K₂HPO₄, 0.1 g/LMgSO₄.7H₂O, 10 g/L defatted soy flour, and 0.1 g/L antifoaming agent.The initial pH of the seed culture was adjusted to 7.0 by adding 1 M HClor NaOH.

The 500 mL flask culture was transferred into a first stage seedfermentor of about 10 L to 20 L that contained the seed culture medium,and was grown therein at 37° C. for 8 hours to reach 2.5% to 5% inoculumsize. The first stage seed culture was then transferred to a secondstage seed fermentor of 100 L, 250 L or 800 L, and was grown therein at37° C. for 8 hours.

Production Media. The production culture medium used for B.licheniformis strain T1 contained 0.7 g/L KH₂PO₄, 1.4 g/L K₂HPO₄, 0.1g/L MgSO₄.7H₂O, 13 g/L defatted soy flour, 40 g/L starch, 13 g/L feathermeal, and 0.1 g/L antifoaming agent. The initial pH of the productionculture was adjusted to 7.0 by adding 1 M HCl or NaOH.

The second stage seed culture was transferred to a production fermentorthat contained the production culture medium for final stage culturing.The final stage culture was carried out at 37° C. for 48 hours beforeharvesting.

During the above culturing steps, the initial pH of the culture mediumwas adjusted to 7.0, but no pH control was provided. The optimal levelof dissolved oxygen was about 30% for B. licheniformis strain T1. Theinoculum size was about 2.5 to 5%, and the inoculum age was about 12hours.

Recovery and Downstream Processing. Enzyme activity in the productionculture was checked before harvesting. The culture supernatant wasseparated from the cell mass via centrifugation, and then concentratedvia ultrafiltration or evaporation. The concentrated liquid enzyme wasthen spray-dried.

Alternatively, the culture supernatant was directly spray-dried afterseparation from the cell mass, without being concentrated.

Enzyme Yield and Enzyme Activity. For 100 L production culture, theenzyme activity measured by azocasein assay before harvesting was 30,000to 35,000 U/mL, and the cell number was 6×10⁹ CFU/mL. The total dryweight of the 100 L production culture was 40 g/L, including 15 g/Linsoluble dry weight and 25 g/L soluble dry weight.

The crude enzyme yield from the directly dried culture supernatant was20 g/L, while the crude enzyme yield from a culture concentrate, asobtained via Pellicon filtration with a 10 kDa molecular weight cut-off,was 16 g/L. The enzyme activity of the crude dry enzyme was greater than1,000,000 U/g, as measured by the azocasein assay.

EXAMPLE 3 Materials and Methods of Supplementation of Poultry Feed withKeratinase

Birds and Housing. Three experiments were conducted. In each experiment,192 day-old broiler chicks were weighed and randomly assigned to 24cage-pens in a completely randomized design to two Alternate Designbatteries (Wilveco, Billerica, Mass.). Birds were weighed, wing-bandedand introduced to the experimental treatments at five (experiments oneand two) or one (experiment three) day of age. Each treatment wasreplicated five times with eight birds per pen except for the controltreatment that was replicated four times with eight birds per pen. Thebirds were housed in a room with controlled temperature, ventilation,and lighting (24 hours/day). During the experimental period the birdswere feed ad libitum in trough feeders and water by nipple drinkers.

The PWD-1 Keratinase Enzyme. The enzyme, PWD-1 keratinase was producedwith a 150-L fermentor using standard methods (Wang and Shih (1998) J.Indust. Microb. Biotech. 22:608-616). Briefly, Bacillus licheniformisPWD-1 (Williams, et al. (1990) supra) was grown in the fermentor at 50°C. for 48 hours. The cell-free media were concentrated by membraneultrafiltration and dried by a freeze-dryer. Typically, the yield of thecrude enzyme was 2.0 g/L. The crude keratinase had an activity of300,000 U/g as measured by the hydrolysis of azo-keratin (Lin, et al.(1992) supra).

DietaryTreatments. All diets were formulated using least-cost linearprogramming software and are presented in Table 1.

TABLE 1 Dietary treatment Ingredient High protein¹ Control² Low ProteinCorn 49.60 59.00 49.00 Soybean meal, 48% CP 41.44 32.00 26.60 Limestone1.32 1.40 1.32 Dical phosphate 1.75 1.70 1.82 Poultry fat 5.34 5.00 4.20DL-Methionine 0.15 0.16 0.13 Salt 0.40 0.50 0.42 Choline chloride 0.100.10 0.08 Minerals³ (TM-90) 0.12 0.12 0.10 Vitamins⁴ (NCSU-90) 0.07 0.070.06 Selenium premix⁵ 0.07 0.07 0.06 Sodium Bicarbonate 0.10 0.10 0.08Starch, corn 0 0 16.60 Total (kg) 100.46 100.22 100.47 Anal- Analysis⁶ %NRC ysis % NRC Analysis % NRC Crude 25.00 108.70 20.23 88.00 16.80 72.90protein, % ME, kcal/kg 3,050 95.31 3,201 100.00 3,257 101.80 Met + Cys,0.933 103.67 0.85 99.00 0.70 77.90 % Lysine, % 1.437 130.60 1.17 106.300.97 88.10 Calcium, % 1.00 100.00 1.03 103.30 1.01 100.10 Available 0.45100.00 0.45 100.70 0.45 100.70 Phosphate, % ¹Provided to birds adlibitum for the first 5 days of age in experiment three only. ²Providedto birds ad libitum for the first 5 days of age in experiments one andtwo. In all experiments, birds on the control treatment continued toreceive the same diet after the first 5 days of age while other birdswere subjected to corresponding treatments. ³The mineral premix wasobtained from Eastern Minerals, Inc., Henderson, NC and provided thefollowing (per kg of diet): 120 mg Zn from ZnSO₄; 120 mg Mn from MnSO₄;80 mg Fe from FeSO₄C5H₂O; 10 mg Cu from CuSO₄; 2.5 mg I from CaIO₄; and1 mg Co from CoSO₄. ⁴The vitamin premix was obtained from Roche, Nutley,NJ and provided the following (per kg of diet): 13,200 IU vitamin A;4,000 ICU vitamin D; 66 IU vitamin E; 39.6 Fg vitamin B₁₂; 13.2 mgriboflavin; 110 mg niacin; 22 mg d-pantothenate; 0.4 mg vitamin K; 2.2mg folic acid; 4.0 mg thiamin; 7.9 mg pyridoxine; 0.253 mg biotin; 100mg ethoxyquin. ⁵The selenium premix provided 0.2 mg Se/kg diet asNa₂SeO₃. ⁶Calculated analysis.

All feed was fed in mash form throughout the experiments. Birds receiveda basal diet at one day of age and were subsequently switched to thecorresponding experimental diets at five days of age. In experiment one,the basal diet fed for the first five days was about 93% of the NationalResearch Council's (NRC) recommendations for crude protein ((1994)supra) but provided 100% for essential amino acids, energy, and calciumand phosphorus contents. In experiments two and three, the basal dietfed for the first 5 days was about 95% of the NRC recommendations((1994) supra) for energy and 100% of the calcium and phosphorus butprovided 105% crude protein content. Subsequently, a pen of broilerchicks was subjected to one of five dietary treatments through to theend of each experiment (21 days in experiments one and three, 26 days inexperiment two). The five dietary treatments in experiment one and twowere: 1) unsupplemented control diet (C, 21.39% crude protein); 2) lowprotein diet (LP, 18% crude protein); 3) low protein diet supplementedwith 0.05% (wt/wt) enzyme preparation (LP+0.05E); 4) low protein dietsupplemented with 0.10% (wt/wt) enzyme preparation (LP+0.10E); and 5)low protein diet supplemented with 0.15% (wt/wt) enzyme preparation(LP+0.15E). The control diet was the same basal diet fed to the birdsfor the first five days of age in experiment one. Birds on treatment onecontinued to receive the same diet after five days of age, while therest of the treatments were switched to the experimental diets at fivedays of age. The dietary treatments in experiment three were: 1)unsupplemented control (C, 21.39% crude protein); 2) control dietsupplemented with 0.10% (wt/wt) enzyme preparation (C+0.10E); 3) lowprotein diet (LP, 18% crude protein); 4) low protein diet supplementedwith 0.10% (wt/wt) enzyme preparation (LP+0.10E); and 5) same astreatment two but fed to birds starting at one day of age rather thanfive days of age.

The enzyme dosage was dissolved in 0.10 N sodium carbonate solution in aratio of 1 gram enzyme/10 ml solution prior to feed application.Thereafter, the enzyme solution was sprayed on top of the feed using aspray bottle in a ratio of 10 ml enzyme preparation/kg diet and mixedusing a small bowl mixer (The Hobart Manufacturing is Company, Troy,Ohio).

Viscosity of Digestae. At the end of each experiment, all of the birdsbut two per pen were euthanized using CO₂ gas. The two remaining birdsper pen were kept for the next day. Early the next day, feeders wereremoved from the pens to produce a 2-hour period with no access to feed.After the 2 hours, birds were once again allowed access to the feed adlibitum. An hour later, the birds were removed, two at a time, and weresubsequently euthanized using CO₂ gas. Necropsy was carried outimmediately after euthanization, and the jejunal contents were emptiedinto 1-ml Eppendorf tubes. Two samples were obtained per bird. The tubeswere immediately centrifuged at 12,000×g for 5 minutes and immediatelyplaced in ice until viscosity was measured using a commercial typeviscometer (Brookfield Digital Viscometer, Model DV-II Version 2.0,Brookfield Engineering Laboratories, Inc., Stoughton, Mass.). Viscosityreading was conducted under conditions that avoided any bacterial growthin the solution.

Data Analysis. Body weights and feed consumption were recorded at 5-dayintervals starting day one of age and through the end of eachexperiment. Feed conversion ratio (feed-to-weight gain), corrected formortality and culls, was calculated. Mortality was recorded daily. Bodyweight, feed consumption, feed conversion ratio, and viscosity readingsof each experiment were analyzed separately using one-way analysis ofthe general linear model procedures of SAS software (SAS Institute(1996) SAS/STAT User's Guide: Statistics, Release 6.11. SAS Institute,Inc., Cary, N.C.). The percentage data were subjected to ANOVA afterarcsine square root percentage transformation. The means were separatedusing least significant difference. Statements of significance werebased on P≦0.05.

EXAMPLE 4 Supplementation of Poultry Feed with Keratinase: Experiment 1

Final body weight, cumulative feed consumption, and feed conversionratios are presented in Table 2.

TABLE 2 Body Weight Feed Feed Conversion Treatment (g) Consumption (g)ratio Control (C) 709 ± 16  901^(a) ± 26 1.56 ± 0.05 Low protein (LP)668 ± 14 835^(ab) ± 23 1.57 ± 0.04 LP + 0.05 E 691 ± 14 860^(ab) ± 231.56 ± 0.04 LP + 0.10 E 700 ± 14 847^(ab) ± 23 1.51 ± 0.04 LP + 0.15 E677 ± 14  826^(b) ± 23 1.54 ± 0.04 ^(a,b)Means within a column withdiffering superscripts differ significantly (P < 0.05) according to theleast squares means function of SAS software (SAS Institute (1996)supra). ¹Values represent means of four to five pens of eight broilerchicks per pen. Values represent means ± standard error of the mean. ²E= enzyme.

The enzyme treatments generally improved body weight with a probabilityvalue of P>0.05. The low protein+0.10% enzyme treatment had higher bodyweight than the low protein treatment (700 vs. 668 grams for lowprotein+0.10% enzyme vs. low protein, respectively, P=0.08). The lowprotein+0.10% enzyme treatment had the highest body weight among theenzyme treatments and was not different from the control treatment (700vs. 709 grams for the low protein+0.10% enzyme vs. control,respectively).

There were no significant differences among the treatments in feedconsumption except between the low protein+0.15% enzyme and the controltreatment (826 vs. 901 grams for the low protein+0.15% enzyme vs.control, respectively, P<0.05). There were no significant differences infeed consumption among the enzyme treatment groups.

There was only one dead bird throughout the whole experiment. The weightof this dead bird and the culls were included in the calculation of feedconversion ratio, which is presented in Table 2. Dietary enzymesupplementation had marginal effects on feed conversion ratio. On acumulative basis, the low protein+0.10% enzyme treatment had the lowestfeed conversion ratio (1.51 vs. 1.57 for the low protein+0.10% enzymevs. low protein, respectively, P>0.05).

The results of the first experiment revealed a trend in the response forthe enzyme treatment. To further analyze the positive effect of theenzyme for a longer time, a second experiment was conducted growing thebirds 5 more days (to 26 days of age).

EXAMPLE 5 Supplementation of Poultry Feed with Keratinase: Experiment 2

This experiment was a repeat of experiment 1 except that birds weregrown 5 more days (to 26 days of age). Final body weight, cumulativefeed consumption, and feed conversion ratio are presented in Table 3.

TABLE 3 Body Feed Feed Conversion Treatment Weight (g) Consumption (g)ratio Control (C) 1089^(a) ± 15 1717^(c) ± 16 1.83^(a) ± 0.04 Lowprotein (LP)  964^(c) ± 13 1734^(b) ± 14 2.14^(c) ± 0.04 LP + 0.05 E1019^(b) ± 13 1796^(a) ± 14 2.08^(bc) ± 0.04  LP + 0.10 E 1025^(b) ± 131764^(ab) ± 14  2.02^(b) ± 0.04 LP + 0.15 E 1032^(b) ± 13 1794^(a) ± 142.04^(bc) ± 0.04  ^(a,b,c)Means within a column with differingsuperscripts differ significantly (P < 0.05) according to the leastsquares means function of SAS software (SAS Institute (1996) supra).¹Values represent means of four to five pens of eight broiler chicks perpen. Values represent means ± standard error of the mean. ²E = enzyme.

There was an improvement in body weight (P<0.05) upon supplementing thelow protein diet with all three levels of the enzyme at 26 days of age.The low protein+0.10% enzyme treatment and the low protein+0.15% enzymetreatment gave the highest body weight improvement (1,032 and 1,025 vs.964 grams for the low protein+0.15% enzyme and low protein+0.10 vs, lowprotein, respectively, P<0.05). However, all the enzyme treatments hadlower (P<0.05) body weight than the control treatment (1,032, 1,025, and1,016 vs. 1,089 grams for the low protein+0.15% enzyme, lowprotein+0.10% enzyme and the low protein+0.05% enzyme vs. control,respectively).

All birds receiving the low protein diet consumed more feed than thecontrol group (P<0.05). Enzyme treatment groups also consumed more feedthan the low protein diet (P<0.05; Table 2). There were no significantdifferences in feed consumption among the enzyme treatments. Enzymesupplementation at 0.05 and 0.15% levels resulted in numerically betterfeed conversion ratios than the low protein treatment, while the lowprotein+0.10% enzyme treatment showed significantly (P<0.05) a betterfeed conversion ratio than the low protein treatment (2.02 vs. 2.14 forthe low protein+0.10% enzyme vs. low protein, respectively). In thisexperiment, supplementing the low protein diet with enzyme did notimprove the performance of the chicks to a level equivalent to that ofthe control diet. However, supplementing the low protein diet with the0.10% enzyme (wt/wt) level did (P<0.05) improve the performance ofchicks over that of the low protein diet.

The birds in both experiments 1 and 2 were provided the control diet forthe first 5 days of age prior to being subjected to the treatment diets.Although the control diet provided adequate energy, calcium andphosphorus, and essential amino acids, it provided only 93% of the NRC((1994) supra) crude protein recommendation, which made it marginallyadequate and sensitive to protease supplementation.

EXAMPLE 6

Supplementation of Poultry Feed with Keratinase: Experiment 3 Inexperiment 3, birds were provided a high protein prestarter dietproviding 105% of the NRC ((1994) supra) recommendation for crudeprotein and slightly higher than requirements for all other nutrientsexcept for energy (95% of NRC recommendations; Table 1). Experiment 3was conducted to determine whether or not the enzyme would continue toexert its effect even after the chicks had received adequate nutrientrequirements.

In this experiment, only one level of the enzyme was used (0.10% wt/wt).However, two new treatments were introduced to test the enzyme's abilityto exert an effect upon supplementation to marginally adequate broilerstarter diets. The two new treatments consisted of supplementing thesame control diet (21.39% crude protein) used in experiments 1 and 2with 0.10% enzyme (wt/wt) and introducing the treated feed to the chickseither at five days (treatment 2) or one day (treatment 5) of age. Thisprovided information whether enzyme supplementation at one day of agewould have any further improvements in performance.

Final body weight, cumulative feed consumption, and feed conversionratio are presented in Table 4.

TABLE 4 Body Weight Feed Feed Conversion Treatment (g) Consumption (g)ratio Control (C) 695^(b) ± 14  974^(b) ± 18 1.49^(ab) ± 0.03  C + 0.10E 767^(a) ± 13 1046^(a) ± 16 1.45^(a) ± 0.03 Low protein (LP) 651^(c) ±13 1043^(a) ± 16 1.71^(c) ± 0.03 LP + 0.10% E 679^(bc) ± 13   978^(b) ±16 1.53^(b) ± 0.03 C + 0.10% E³ 764^(a) ± 13 1022^(ab) ± 16  1.42^(a) ±0.03 ^(a,b,c)Means within a column with differing superscripts differsignificantly (P < 0.05) according to the least squares means functionof SAS software (SAS Institute (1996) supra). ¹Values represent means offour to five pens of eight broiler chicks per pen. Values representmeans ± standard error of the mean. ²E = enzyme. ³The enzyme was addedat one day of age in this treatment. All others were added at five daysof age.

Although the data in Table 2, Table 3, and Table 4 show only the finalbody weight numbers of the birds in the different experiments, the birdswere weighed every 5 days in each of the experiments. Looking at the5-day interval numbers for this experiment, it was clear thatsupplementing the low protein diet with the enzyme in experiment 3showed similar effect on body weight gain to those of experiments 1 and2. Supplementing the low protein diet with the enzyme increased the21-day body weight of the birds, but the effect could not be detected atP<0.05 (679 vs. 651 grams for the low protein+0.10% enzyme vs, lowprotein, respectively, P>0.05). However, supplementing the control dietwith the enzyme (control+0.10% enzyme preparation, treatments 2 and 5)showed higher body weight than the control treatment (767 and 764 vs.695 grams for treatments 2 and 5 vs, control, respectively, P<0.05).Unexpectedly, supplementation of the control diet with the enzyme showedsignificantly higher improvements in body weight than when the lowprotein diet was supplemented with the enzyme whether the enzyme wassupplemented at one or five days of age (Table 4). This may have beendue to the higher protein and/or amino acids content of the control dietvs. the low protein diet. Keratinase is a broad-spectrum protease enzymethat attacks proteins of different sources and breaks them down intosmaller polypeptide components. These polypeptides become easier todegrade by the digestive enzymes in the lumen of the intestines. Highercrude protein and/or amino acids content of the diet (in this case thecontrol diet) means higher substrate content for the enzyme to work onreleasing more protein components and making it more available to theyoung chick which, in turn, will be reflected in higher body weightgain.

EXAMPLE 7 Supplementation of Poultry Feed with Keratinase: DigestaeViscosity

The viscosity readings (mPas) of the jejunal contents of 22-day-old(experiments 1 and 2) and 27-day-old (experiment 3) broiler chicks fromall three experiments are presented in Table 5.

TABLE 5 Treatment Experiment 1 Experiment 2 Experiment 3 Control (C)3.65^(b) ± 0.42 2.31^(b) ± 0.15 2.55^(ab) ± 0.17  C + 0.10 E — —2.18^(b) ± 0.15 C + 0.10 E³ — — 1.99^(b) ± 0.15 Low protein (LP)3.59^(b) ± 0.38 2.36^(ab) ± 0.14  2.97^(a) ± 0.15 LP + 0.05 E 2.98^(ab)± 0.38  2.78^(a) ± 0.14 — LP + 0.10 E 2.88^(ab) ± 0.38  2.21^(bc) ±0.14  2.20^(b) ± 0.15 LP + 0.15 E 2.27^(a) ± 0.38 1.98^(c) ± 0.14 —^(a,b,c)Means within a column with differing superscripts differsignificantly (P < 0.05) according to the least squares means functionof SAS software (SAS Institute (1996) supra). ¹Values represent means offour to five pens (16-20 numbers). Values represent means ± standarderror of the mean. ²E = enzyme. ³The enzyme was added at one day of agein this treatment. All others were added at five days of age.

Supplementing both the low protein and the control diets in all theexperiments with keratinase reduced the viscosity of jejunal contents.The reduction was directly proportional to the level of enzymesupplementation. Supplementing the low protein diet with 0.15% enzyme(low protein+0.15% enzyme) reduced the viscosity of the jejunal contentsin experiments 1 and 2 (2.27 and 1.98 mPas vs. 3.59 and 2.36 mPas forlow protein+0.15% enzyme vs. low protein in experiments 1 and 2,respectively, P<0.05). The low protein+0.15% enzyme treatment also had alower jejunal viscosity when compared to the control treatment (2.27 and1.98 mPas vs. 3.65 and 2.31 mPas for the low protein+0.15% enzyme vs.control in experiments 1 and 2, respectively, P<0.05).

When supplementing the control diet with keratinase at 5 days of age,jejunal viscosity was also reduced (2.18 mPas vs. 2.55 mPas for thecontrol+0.10% enzyme [experiment 3, treatment 2] vs. control,respectively, P>0.05). However, the reduction was significant only whenthe diet supplemented with the enzyme starting at 1 day of age (1.99mPas vs. 2.55 mPas for the control+0.10% enzyme [experiment 3, treatment5] vs. control, respectively, P<0.05).

EXAMPLE 8 Feeding Trials Using Varying Enzymatic Activity

A dry crude enzyme extract produced according to methods describedherein is composed primarily of keratinase enzyme, but may also containother types of compounds, including other enzymes, carbohydrates,non-enzymatic peptides, nucleotide fragments, etc., which have amolecular weight of greater than 5 kDa and are therefore retained uponultrafiltration.

Additional experiments were conducted to study the correlation betweenthe growth performance of broiler chicks and the keratinase activity ofthe crude enzyme extracts added to their diets.

Crude enzyme extracts obtained from fermentation production of B.licheniformis strains P1, P2, and T399 were used in these studies andthe results are provided in Table 1.

TABLE 1 Enzyme Crude Enzyme Activity Inclusion Trial # Extract Type(Unit/g) Rate (%) Growth Performance MT P1 strain 1,000,000 0.05 Noeffect 301 (in 0.5% soy flour)¹ 0.10 Improvement (0.05 < P < 0.1) 0.15Improvement (0.05 < P < 0.1) MT P1 strain 1,000,000 0.05 Significantimprovement in 401 (in 0.5% soy flour)² low protein 0.10 Significantimprovement in low protein 0.15 Significant improvement in low proteinMT P1 strain 1,000,000 0.10 Significant improvement in 501¹ (in 0.5% soyflour)¹ low protein and control MT P1 strain 300,000 0.25 Negativeeffect 701 (in 0.5% soy flour)¹ P2 strain 450,000 0.17 Slightimprovement (in 0.5% soy flour)¹ MT P1 strain (inhibited 300,000 0.25 Noeffect 801 by PMSF)³ P1 strain 300,000 0.25 No effect (inactivated byheat)⁴ MT P2 strain 450,000 0.10 Significant improvement 901 (in 1% soyflour)¹ P2 strain 800,000 0.10 No effect (in 2%, 1% soy flour)¹ T399strain <5000 0.10 No effect (in 2%, 1% soy flour)¹ KE P2 strain 600,0000.10 Improvement in 23% diets 202 (in 1% soy flour)¹ P2 strain 600,0000.10 Improvement in 21 & 23% (in 2% soy flour)¹ diets KE P1 strain⁵150,000 0.10 Significant improvement in all 302 Pilot P1 strain¹ 300,0000.10 Improvement in 21% diet Test P1 strain¹ 300,000 0.25 Improvement in21% diet ¹Enzyme supplement fed from 6 to 21 days of age. ²Enzymesupplement fed from 6 to 27 days of age. ³>90% enzymatic activity isinhibited. ⁴>98% enzymatic activity is inhibited. ⁵Enzyme supplement fedfrom 1 to 21 days of age.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is describedby the following claims, with equivalents of the claims to be includedtherein.

1-28. (canceled)
 29. A method of growing meat poultry, comprising:feeding said meat poultry a feed comprising (a) soybean meal and sorghumas a poultry diet, wherein said poultry diet is essentially free ofkeratin, and (b) Bacillus licheniformis PWD-1 keratinase in an amounteffective to enhance the weight gain of said meat poultry.
 30. Themethod of claim 29, wherein the meat poultry is an immature bird. 31.The method of claim 29, wherein the meat poultry is a chicken, turkey orduck.
 32. The method of claim 31, wherein the chicken is from about 1day to 65 days old.
 33. The method of claim 31, wherein the chicken isfrom about 1 day to 21 days old.
 34. The method of claim 31, wherein thechicken is from about 1 day to 7 days old.
 35. The method of claim 31,wherein the chicken is a broiler chick.
 36. The method of claim 29,wherein the poultry diet is a starter diet.
 37. The method of claim 29,wherein the poultry diet is a grower diet.
 38. The method of claim 29,wherein the poultry diet is a finisher diet.
 39. The method of claim 29,wherein the poultry diet further comprises at least one of corn, oats,barley, wheat, triticale and lye.
 40. The method of claim 29, whereinthe poultry diet further comprises at least one of corn, barley, wheatand rye.
 41. The method of claim 29, wherein the poultry diet furthercomprises corn.
 42. The method of claim 29, wherein the Bacilluslicheniformis PWD-1 keratinase is a crude extract or pure enzyme.
 43. Amethod of supplementing a poultry diet of a meat poultry, the methodcomprising: feeding said meat poultry a feed comprising (a) soybean mealand sorghum, and (b) Bacillus licheniformis PWD-1 keratinase in anamount effective to enhance the weight gain of said meat poultry,wherein said feed is essentially free of keratin.
 44. A method ofimproving the efficiency of feed utilization of an animal feed in meatpoultry, comprising: feeding said meat poultry a feed comprising (a)soybean meal and sorghum as a poultry diet, wherein said poultry diet isessentially free of keratin, and (b) Bacillus licheniformis PWD-1keratinase in an amount effective to improve the efficiency of feedutilization of an animal feed in said meat poultry.
 45. A method ofincreasing the digestibility of an animal feed in meat poultry,comprising: feeding said meat poultry a feed comprising (a) soybean mealand sorghum as a poultry diet, wherein said poultry diet is essentiallyfree of keratin, and (b) Bacillus licheniformis PWD-1 keratinase in anamount effective to increase the digestibility of an animal feed in saidmeat poultry.
 46. A method of reducing mortality in meat poultry,comprising: feeding said meat poultry a feed comprising (a) soybean mealand sorghum as a starter diet, wherein said poultry diet is essentiallyfree of keratin, and (b) Bacillus licheniformis PWD-1 keratinase in anamount effective to reduce mortality of said meat poultry.
 47. Themethod of claim 46, wherein the meat poultry is an immature bird. 48.The method of claim 46, wherein the meat poultry is a broiler chick. 49.An animal feed comprising soybean meal, sorghum, and Bacilluslicheniformis PWD-1 keratinase, wherein said animal feed is essentiallyfree of keratin.
 50. The animal feed of claim 49, wherein the animalfeed further comprises at least one of corn, oats, barley, wheat,triticale and rye.
 51. The animal feed of claim 49, wherein the animalfeed further comprises at least one of corn, barley, wheat and rye. 52.The animal feed of claim 49, wherein the keratinase is a crude extractor pure enzyme.
 53. The animal feed of claim 49, wherein the animal feedis added to a starter diet, wherein said starter diet is essentiallyfree of keratin.
 54. The animal feed of claim 53, wherein the starterdiet is a soybean meal and sorghum starter diet.
 55. The animal feed ofclaim 49, wherein the animal feed is added to a grower diet, whereinsaid grower diet is essentially free of keratin.
 56. The animal feed ofclaim 49, wherein the animal feed is added to a finisher diet, whereinsaid finisher diet is essentially free of keratin.
 57. A method ofgrowing meat poultry, comprising: feeding said meat poultry a feedcomprising soybean meal and sorghum in mash form as a poultry diet,wherein said poultry diet is essentially free of keratin, and Bacilluslicheniformis PWD-1 keratinase in an amount effective to enhance theweight gain of said meat poultry.
 58. The method of claim 57, whereinthe meat poultry is a chicken, turkey or duck.
 59. The method of claim57, wherein the Bacillus licheniformis PWD-1 keratinase is a crudeextract or pure enzyme.
 60. An animal feed in mash form comprisingsoybean meal, sorghum and Bacillus licheniformis PWD-1 keratinase,wherein said animal feed is essentially free of keratin.
 61. The animalfeed of claim 60, wherein the keratinase is a crude extract or pureenzyme.